Lymphokine activated effector cells for antibody-dependent cellular cytotoxicity (ADCC) treatment of cancer and other diseases

ABSTRACT

This invention relates to processes and compositions for the immunotherapeutic treatment of cancer and non-malignant tumors. More particularly, this invention relates to processes and compositions for enhancing the body&#39;s immune response by increasing the cytotoxic activity of cells which mediate antibody dependent cellular cytotoxicity. Cells which are characterized by increased cytotoxic activity, as a result of the process of this invention, are useful in methods and compositions for the treatment of various types of cancer and non-malignant tumors.

This application is a continuation of Ser. No. 07/808,958, filed Dec.13, 1991, now U.S. Pat. No. 5,308,626, which is a continuation of Ser.No. 07/355,148, filed May 16, 1989, now abandoned, which is acontinuation of Ser. No. 07/050,292, filed Apr. 27, 1987, now abandoned,which is a continuation-in-part of Ser. No. 750,091, filed Jun. 28,1985, now abandoned.

TECHNICAL FIELD OF INVENTION

This invention relates to the immunotherapeutic treatment of cancer andnon-malignant tumors. More particularly, this invention relates to aprocess for enhancing the body's immune response by increasing thecytotoxic activity of cells which mediate antibody dependent cellularcytotoxicity. Cells which are characterized by increased cytotoxicactivity, as a result of the process of this invention, are useful inmethods and compositions for the treatment of various types of cancerand non-malignant tumors.

BACKGROUND ART

The transformation of normal cells within the body into cancerous andnon-malignant neoplasms may be induced by viral infections, chemicalcarcinogens, radiation, physical agents or spontaneous tumorigenicgrowth. As a result of such transformation, normal cell surface antigenexpression may be altered. New antigens--tumor specific antigens orantigens characteristic of premature cell types--or antigen expressionpattern changes may be demonstrated on many tumor types. These antigenicchanges are targets for the body's immune response.

In some instances, the extent or rate of cell transformation may exceedthe capabilities of the body's immune response. Alternatively, theimmune response may itself be ineffective or deficient. Supplementalmethods have, therefore, been used in the treatment of transformedcells. These methods include non-surgical treatments, such aschemotherapy and radiation, and surgical treatments. Typically, thesetreatments are characterized by a range of undesirable side effects.Non-surgical treatments having immunosuppressant effects may increasethe patient's susceptibility to secondary infections. Surgicaltreatments to excise transformed cells involve risks attendant withinvasive procedures and may not effectively remove or eliminate theentire transformed cell population.

An alternative method of treatment for cancers and non-malignant tumorshas involved the use of monoclonal antibodies to tumor specific antigenson the surface of transformed cells. The effectiveness of suchtreatments, typically involving murine monoclonal antibodies, is oftenlimited by a variety of factors. For example, human patients treatedwith murine monoclonal antibodies may develop an anti-murineimmunoglobulin response which severely reduces the effectiveness offurther administration of murine monoclonal antibodies (G. E. Goodman etal., "Pilot Trial of Murine Monoclonal Antibodies In Patients WithAdvanced Melanoma", Journal Of Clinical Oncology, 3, pp. 340-51 (1985)).Other reported side effects of monoclonal antibody treatments includeanaphylaxis, fever and chills.

Monoclonal antibodies are also ineffective in the treatment of thosetumor specific surface antigens which modulate upon exposure to specificantibodies. Such antigens include, for example, the common acutelymphocytic leukemia antigen ("CALLA"), which appears on the surface oftransformed cells of a majority of patients suffering from acutelymphocytic leukemia (J. Ritz et al., "Serotherapy Of AcuteLymphoblastic Leukemia With Monoclonal Antibody", Blood, 58, pp. 141-52(1981)). When the CALLA antigen on the surface of a tumor cell isexposed to its specific antibody, the antigen migrates and the cell mayinternalize the antigen and antibody, preventing recognition of thetumor cell as a target by immune response effector cells such asleukocytes, lymphocytes, macrophages, killer ("K") cells or naturalkiller ("NK") cells.

In view of the disadvantages of such supplemental treatments, varioustherapies have been directed to augmenting the body's natural immuneresponse to transformed cells. It is known that in the presence ofantibodies, certain effector cells, such as lymphoid cells havingsurface bound receptors for the Fc regions of antibodies, mediate anantibody dependent cellular cytoxicity ("ADCC") reaction against targetcells. By means of ADCC, these effector cells exert cytolytic activityagainst such target cells.

Two types of ADCC reactions have been demonstrated in vitro. Inclassical ADCC reactions, effector cells attach to antibody-coatedtarget cells and subsequently cause cytolysis of the target cells (A. H.Greenberg et al., "Characteristics Of The Effector Cells MediatingCytotoxicity Against Antibody-Coated Target Cells. I., Immunology, 21,p. 719 (1975)). This attachment between effector and target cell resultsfrom the interaction of the Fc region of the antibody coating the targetcell and the Fc receptor of the effector cell. One disadvantage of thistype of ADCC reaction is that it may be hampered by circulatingantigen-antibody complexes, often associated with various diseases,which compete with the target-cell bound antibody for the Fc receptorsof the effector cells (I. C. M. MacLennan, "Competition For ReceptorsFor Immunoglobulin On Cytotoxic Lymphocytes", Clin. Exp. Immunol., 10,p. 275 (1972)). Due to this drawback of classical ADCC, a second type ofADCC reaction--antibody-directed ADCC--has been proposed. Inantibody-directed ADCC, the target-specific antibody is first attachedto the effector cell and the resulting complex is then "directed", viathe antibody, to its specific antigen on the target cell surface.Advantageously, antibody-directed ADCC may not be affected by thepresence of antigen-antibody complexes circulating in the host system.

The interaction between antibodies and effector cells via Fc region/Fcreceptor attachment is normally weak. And, in some instances, antibodiesdo not remain associated with effector cells for a period of timesufficient to permit lysis of target cells. In view of this potentialproblem, antibodies have been attached to the effector cells usingpre-treatment with polyethylene glycol and a mixture of phthalate oils(J. F. Jones and D. M. Segal, "Antibody-Dependent Cell MediatedCytolysis (ADCC) With Antibody-Coated Effectors: New Methods ForEnhancing Antibody Binding And Cytolysis", J. Immunol., 125, pp. 926-33(1980)). The applicability of this method for in vivo treatments,however, may be diminished by the toxic effects that any polyethyleneglycol and phthalate oil residues on the antibody-effector cell complexmay have on the body.

Alternatively, a method has been proposed for enhancingantibody-directed ADCC by adjuvant chemotherapy with cytotoxic drugs (I.R. Mackay et al., "Effect On Natural Killer And Antibody-DependentCellular Cytotoxicity Of Adjuvant Cytotoxic Chemotherapy IncludingMelphalan In Breast Cancer", Cancer Immunol. Immunother., 16, pp. 98-100(1983)). Such a method, however, risks undesirable side effectsresulting from the use of cytotoxic drugs.

Therefore, conventional means for treating cancer and non-malignanttumors by either supplementing or enhancing the body's immune responseare characterized by various disadvantages. The need, thus, exists foran effective process and composition which avoid those disadvantageswhile providing effective treatment for cancers and non-malignanttumors.

DISCLOSURE OF THE INVENTION

The present invention solves the problems referred to above by providinga process for enhancing the immune response of the body to cancers andtumors by activating effector cells which mediate antibody dependentcellular cytotoxicity ("ADCC") to increase their cytotoxic activitybeyond normal levels. Effector cells which are characterized by suchincreased cytotoxic activity, as a result of this process, may thenadvantageously be used in methods and compositions of this invention forthe immunotherapeutic treatment of cancer and non-malignant tumors. Inaddition, one preferred embodiment this invention provides a process formore efficiently directing these activated effector cells to targetcells by the attachment of target-specific antibodies to the Fcreceptors on the surface of the activated effector cells. It alsoprovides compositions characterized by such specific antibody effectorcell complexes.

The processes and compositions of this invention may be used to causeregression, remission or prevention of transformed cell growth, toprevent metastatic growth throughout the body or to increase immunity ofthe host upon rechallenge with the disease-causing agent.Advantageously, the processes and compositions of this invention enhancethe body's natural immune response of cell-mediated immunity via ADCCand, therefore, do not incur the variety of side effects whichcharacterize conventional cancer and tumor treatments.

DETAILED DESCRIPTION OF THE INVENTION

In a first embodiment, this invention provides a process forpre-selection of Fc receptor positive cells from a pool of bulk lymphoidcells obtained by density gradient centrifugation techniques or othermethods of leukocyte separation.

This invention also relates to a process for treating effector cellswhich mediate antibody dependent cellular cytotoxicity to increase theircytotoxic activity beyond normal levels. One embodiment of this processcomprises the steps of recovering effector cells from a donor andactivating the cells in vitro to increase their antibody dependentcellular cytotoxic activity. This invention also relates to cellsactivated by that process.

This invention also relates to a process and composition for treatingcancer and non-malignant tumors. Generally, this process comprises thestep re-introducing effector cells activated by the processes of thisinvention into the donor or transferring the activated cells to arecipient. According to an alternate and preferred embodiment of thisinvention, antibodies which are specific for the cancer or non-malignanttumor to be treated are first attached to the activated effector cellsbefore they are re-introduced to the donor or transferred to therecipient.

Treatment of effector cells according to the processes of this inventionactivates them by increasing their antibody dependent cytotoxic activityagainst target cells. This increase in antibody dependent cytotoxicactivity ranges between at least about 1.25 and 25 times, and in someinstances between at least about 1.25 and 125 times, that of non-treatedeffector cells in their natural state.

Without being bound by theory, we believe that this increased antibodydependent cytotoxic activity is due to increased Fc receptor expressionon the surfaces of effector cells activated by the processes of thisinvention. Specifically, we believe that our activated cells are betterable to mediate antibody dependent cellular cytotoxicity because more Fcreceptors are present on effector cells after treatment according tothis invention, and therefore tumor-specific antibodies (coating thetarget cells or exogenous to them) having an Fc region are more likelyto attach to the effector cells in an effective way, i.e. via the Fcregion of the antibody to an Fc receptor on the effector cell, so thatthe attachment will be maintained and ADCC ensured.

In addition, since more Fc receptors are present on the activatedeffector cells, a greater number of target-specific antibodies mayattach to each effector cell. The increased density of antibodies pereffector cell not only permits a greater number of antibodies to bepresent on or reach the target transformed cell for each effector, butalso increases the ability of an effector to recognize or locate thetarget cell and, thus, to mediate ADCC. The overall result of theseeffects on treated effector cells is the enhancement of the body'simmune response against target cells. Such enhancement is particularlyadvantageous to the treatment of diseases which cause a decrease in orabsence of effector cells in the patient's immune system.

Diseases which may be treated by the processes and compositions of thisinvention are those characterized by an undesirable proliferation oftarget cells to which antibodies may be specifically directed. Suchdiseases include malignant and non-malignant solid or fluid tumors,carcinomas, myelomas, sarcomas, leukemias and lymphomas. In addition,effector cells activated according to the processes of this inventionmay be administered prophylactically for the prevention of cancer andnon-malignant tumors.

It should also be understood that effector cells activated according tothis invention may be used, preferably with an appropriate antibody, toexert enhanced ADCC activity against any type of target cells. Effectorcells activated according to the processes of this inventiondemonstrated increased cytotoxic activity in ADCC and anti-tumor cellproliferation assays against target human cells such as, for example,those derived from the K562 tumor cell line, a human myelogenousleukemia (H. Pross and M. Baines, "Spontaneous Human Lymphocyte-MediatedCytoxicity Against Tumour Target Cells I. The Effect of MalignantDisease", Intl. J. Cancer, 18, pp. 593-604 (1976)); the Nalm-6 leukemiacell line (gift of Dr. Tucker LeBien, University of Minnesota); freshsamples of acute lymphoblastic leukemia cells and fresh preparations ofchronic myelogenous leukemia cells. Activated effector cells alsodemonstrated increased cytotoxic activity against murine target cellssuch as, for example, Zbtu murine T-cell lymphoma cells; EL-4 murinecells (ATCC Tib-39) P815 murine cells (ATCC Tib-64) and Yac murine cells(R. Kiessling et al., "Genetic Variation Of In Vitro Cytolytic ActivityAnd In Vivo Rejection Potential Of Non-Immunized Semi-Syngeneic MiceAgainst A Mouse Lymphoma Line", Intl. J. Cancer, 15, pp. 933-40 (1975)).

The processes and compositions of this invention may be used to treatany mammal, including humans. Preferably, the source of the effectorcell is the patient to be treated. However, effector-mediated ADCC isneither patient-specific nor species-specific. Effector cells derivedfrom one patient or species and activated according to the processes ofthis invention may, therefore, be used in another patient or species toexert cytotoxic activity against target cells. The use of activatedeffector cells donated from another patient or species may benecessitated for recipients having depressed levels of effector cells.Although such treatment with donated effector cells will ultimately leadto rejection of the cells by the recipient, some degree of antibodydependent cellular cytotoxic activity by the activated effector cellswill normally take place before those cells are rejected and thatactivity may be sufficient to affect the course of the disease to betreated.

Effector Cells

Effector cells which may be activated by the processes of this inventioninclude any type of cytotoxic cell having a surface receptor for the Fcregion of an antibody and which can mediate ADCC. Such effector cellsinclude, for example, leukocytes, such as lymphocytes, monocytes, Kcells and NK cells.

Such cells may be isolated in a wide variety of ways including isolationdirectly from syngeneic donors and from 1 to 7 day cultures of suchcells. For example, effector cells may be isolated by a process in whichspleens were removed from mice, minced and washed in sterile balancedsaline solution and the leukocytes isolated by centrifugation throughlymphocyte separation medium (Lymphopaque, Accurate Chemical andScientific Corporation, Westbury, N.Y.) to a final concentration ofabout 5×10⁷ cells/ml.

Human effector cells may be isolated, for example, from peripheral bloodaccording to a modification of the procedure described by A. Boyum,"Lymphocytes. Isolation, Fractionation and Characterization", J. B.Natvig et al. (Ed.), Scand. J. Immunol., 5: Suppl. 5, pp. 9-15 (1976).In some cases, the leukocyte population may be further sorted bygradient centrifugation (Ficoll-Hypaque density gradient, Pharmacia FineChemicals, Piscataway, N.J.) to separate out the mononuclear whitecells. The mononuclear leukocytes obtained at this point may then beeither used as a source for effector cells or further treated usingstandard techniques to sort out more reactive cells or only those cellshaving Fc receptors. Such techniques include separation or enrichmenttechniques using monoclonal antibodies, sorting with FACS IV(fluorescence activated cell sorter, Becton Dickinson, FACS Division,Sunnyvale, Calif.) or adherence techniques.

For example, various antibodies may be used to obtain the effector cellsto be treated according to the processes of this invention. Antibodiesused to identify, label, characterize, sort, separate, enrich and/ordeplete effector cells treated in the processes of this inventionincluded, for example, the following:

(1) HNK-1, Leu-7 (Becton Dickinson, Mountain View, Calif. - Catalog No.73-90)

(2) Leu-11-a (Becton Dickinson - Catalog No. 75-23)

(3) Leu-11-b (Becton Dickinson - Catalog No. 75-30)

(4) Leu-11-c (Becton Dickinson - Catalog No. 76-17)

(5) Anti-IL2-Receptor (Becton Dickinson - Catalog No. 76-40)

(6) Anti-Human Fc Receptor (New England Nuclear, Boston, Massachusetts -Catalog No. Nei033)

(7) Goat Anti-Mouse IgG Fc Fragment F(ab')₂ (Jackson Immuno-ResearchLabs, Avondale, Pa. - Catalog No. 115-0646)

(8) Rabbit Anti-Goat IgG Fc Fragment F(ab')₂ (Jackson Immuno-ResearchLabs - Catalog No. 305-0608)

(9) Rabbit Antigoat IgG (Cappel, Malvern, Pa. - Catalog No. 0612-3151).

(10) Mouse IgGl (Catalog No. 9041, Litton Bionetics Inc., Kensington,Md.)

(11) MOPC 104E, a purified mouse myeloma protein (Catalog Nol 8402-29,Litton Bionetics, Inc.)

For example, mononuclear leukocytes may be labelled with Anti-Leu-11a,Anti-Leu-11b or Anti-Leu-11c antibodies (Becton Dickinson) and onlythose cells labelled with antibodies may be sorted by positiveselection. Alternatively, since cells that have Fc receptors fall intothe size range of 7μ-25μ diameter, an elutriator centrifuge BeckmanInstruments! which sorts cells based on size may be used.

As an alternative to the above procedures for obtaining effector cellsfrom human donors, it is possible to utilize a leukaphoresis techniqueusing, for example, a Fenwal CS-3000 (Fenwal Labs, Deerfield, Ill.) or aHaemonetics Model 30 leukaphoresis machine (Haemonetics Corp.,Braintree, Mass.) T. Loftus et al., "Leukophoresis: Increasing TheGranulocyte Yield With the Fenwal CS 3000", J. Clin. Apheresis, 1, pp.109-14 (1983) and H. Braine et al., "Peripheral Blood LymphocyteProliferative Response In Vitro And Serum Immunoglobulins In RegularHemaphoresis Donors", J. Clin. Apheresis, 2, pp. 213-18 ((1985)!.Leukophoresis permits leukocytes to be obtained from circulatingperipheral blood via a centrifuge placed in line with a patient accessline. The leukocytes are harvested and red cells are returned to thepatient. This technique advantageously permits the collection of morewhite cells and, therefore, more effector cells having Fc receptors,than those obtained from one unit of blood.

After they are obtained from the patient or donor, effector cells areplaced in a balanced salt solution medium, such as Hanks balanced saltsolution or tissue culture medium such as RPMI-1640 supplemented withheat inactivated 5% fetal calf serum, 5% Ultroser G or 5% autologousserum which may be supplemented with an antibiotic such as, for example,Gentamicin (5 μg/ml), Penicillin (10 units/ml), Streptomycin (10 μg/m),or mixtures thereof, and maintained in a refrigerator at 4° C. If theeffectors are to be maintained in vitro for over 24 hours, they areplaced in a medium which also contains essential nutrients for cellviability. Preferably, they are stored frozen by quickly freezing themto -70° C.

Activation of effector cells according to this invention may be carriedout by treating the cells, in vitro, with lymphokines, allogeneic orxenogeneic serum, or mixtures thereof. The lymphokines used may be thosepurified from natural sources using conventional techniques or thoseproduced by recombinant techniques. The preferred lymphokines for use inthis invention are gamma interferon ("γ-IFN") and interleukin-2("IL-2"), and these may be used alone, in combination, or in series, forthe activation of effector cells. The preferred dosage of γ-IFN is about600 units/ml, with an approximate effective range of between about 200and 2500 units/ml. The preferred dosage of IL-2 is about 750 units/ml,with an approximate effective range of between about 30 and 2000units/ml. Serial combination treatment with these two lymphokines ismost preferably carried out by γ-IFN treatment first, followed by IL-2treatment, at the dosages indicated for the individual treatments.

According to this invention, effector cells are usually treated in vitrowith the lymphokine, allogeneic or xenogeneic serum or mixtures thereofand incubated at 37° C. in a 5% CO₂ atmosphere or refrigerated at 4° C.for a period of time sufficient to permit the cells to be activated sothat their antibody dependent cellular cytotoxicity is increased beyondnormal levels, as determined by serial sampling for verification ofactivated function. More specifically, effector cells in suspensionpreferably having a range of 10⁵ to 10⁷ effector cells per ml of mediaare treated with the lymphokine, allogeneic or xenogeneic serum, ormixtures thereof, for preferably between about 1 and 7 days, mostpreferably between about 3 and 4 days.

Effector cells may be stored frozen after being activated and beforebeing administered to the patient. Alternatively, effector cells may bepartially activated, stored frozen, thawed and further activated beforeuse. For example, activated effector cells may be placed in tissueculture medium supplemented with fetal calf serum, normal human serum,or serum substitutes, such as Ultroser G, and dimethyl sulfoxide("DMSO") and frozen as quickly as possible, to at least -70° C., using acontrolled rate cell freezer or any conventional method for freezing.After storage, frozen activated effector cells may be thawed as quicklyas possible without raising the temperature of the cells above 37° C.

Preferably in the processes and compositions of this invention,antibodies are used with the activated effector cells to mediate anenhanced ADCC effect against target cells and to indicate the level ofeffector cell activation achieved. Such antibodies are those of the IgGclass, preferably IgG2 antibodies, which have Fc regions and whichprimarily recognize antigens that are either specific to a particularcancer or non-malignant tumor or present in higher densities on cells ofcancer or non-malignant tumors than on normal cells.

Examples of such antibodies include the following:

(1) GAGPA-Goat anti-Gross passage A virus (National Institutes OfHealth, Bethesda, Md.)

(2) PAGPA-Pig anti-Gross passage A virus (National Institute Of Health,Bethesda, Md.)

(3) Anti-CALLA (Becton Dickinson, Mountain View, Calif. - Catalog No.7500)

(4) J-5 Anti-CALLA (Coulter Immunology, Florida - Catalog No. 6602143)

(5) BA3 Anti-CALLA (Hybritech, Inc., San Diego, Calif. - Catalog No.0672)

(6) ANTI-HpCA-1 (Becton Dickinson - Catalog No. 7660)

(7) Rabbit Anti-Nalm 6 (a polyclonal antibody raised by hyperimmunizinga New Zealand female rabbit by intravenous injection of 1×10⁷ Nalm-6cells once a week for five weeks).

According to one preferred embodiment of this invention, the antibodies,which are specific, for the target transformed cells, are attached tothe activated effector cells before those cells are administered to thepatient. This attachment may be carried out by incubating the activatedeffector cells in vitro with high concentrations of single or pooledmonoclonal or polyclonal antibodies. For example, this attachment may becarried out by contacting the activated effector cells with antibodies,in a ratio of about 10⁷ cells per 2 mg of antibody and maintaining themixture at room temperature or at 37° C. or refrigerating the mixture at4° C. for between about 1 and 72 hours. Activated effector cells withantibodies attached are termed "armed" effector cells hereinafter.

For example, to attach an antibody to buffy coat leukocytes which havebeen activated according to the processes of this invention, theleukocytes are first washed three times in RPMI-1640 by centrifugationin a Beckman TJ-6 centrifuge equipped with a TH-4 rotor at 1200 rpm for5 minutes. The multistep washing procedure removes loosely boundcytophilic immunoglobulins and may increase the availability of Fcreceptors for binding of the desired antibodies. The effector cells maythen be further purified by centrifugation on a density gradient mediumsuch as Ficoll-Hypaque (Litton Bionetics, Bethesda, Md.). The collectedleukocyte interface may then be resuspended in RPMI-1640 mediumcontaining antibodies at concentrations of at least about 5×10⁷ effectorcells per 0.5 to 2.0 mg antibodies and incubated at room temperature or37° C. or refrigerated at 4° C. for between about 1 and 72 hours withgentle rocking. The effector cells may then be allowed to sedimentthrough gelatin plasma substitutes, such as Plasmagel (HIT Corporation,Buffalo, N.Y.) or by centrifugation on density gradient medium such asFicoll-Hypaque, to "fix" the antibody attachment to the effector cellsprior to administration to the patient. Effector cells attached totarget-specific antibodies may then be administered to the patient asdescribed below.

Patients with cancer or non-malignant tumors may be treated by theadministration of a therapeutically or oncologically effective amount ofeffector cells which have been activated according to the processes ofthis invention. These activated effector cells also preferably haveantibodies specific for the target cells attached to them beforeadministration, as described supra. The activated effector cells may beadministered in liquid dosage forms by means of intravenous injection orinfusion. Injectable/infusable solutions of activated effector cells mayalso include conventional pharmaceutically acceptable carriers such assterile saline solution.

The amount of activated effector cells administered at one time or overa series of treatments, will depend upon the volume and activity of theactivated effector cells available for and present in the initial dose,the patient's health status, the severity and course of the cancer ornon-malignant tumor and the judgment of the treating physician.Effective dosages may be in the range of between about 1×10⁶ and 5×10¹⁰cells. The effectiveness of treatments may be assessed by analyzingsamples of blood or bone marrow (in cases of leukemias and lymphomas) orby measuring rates of reduction on tumor load (in cases of tumorigenicdiseases) at various post-treatment intervals.

When the treated effector cells are to be administered withoutantibodies attached, the patient may be pre-treated with antibodies orother substances to increase the levels of circulating antibodies in thebody, preferably tumor specific ones. For example, the patient may betreated by intravenous injection or infusion with a single monoclonal orpolyclonal tumor-specific antibody or a pool of two or moretumor-specific monoclonal or polyclonal antibodies. Useful antibodiesare immunoglobulin-G "IgG" class antibodies having demonstrable ADCCmediating potentials, as assessed by in vitro analyses, and whichrecognize tumor-specific antigens or antigens found preferentially or ingreater densities on cancerous or non-malignant tumor cells.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLES

In the following examples, two assays were used to assess the cytotoxicactivity and anti-tumor proliferation potentials of effector cellsbefore and after being activated according to the processes of thisinvention. These assays were an ADCC ⁵¹ chromium release assay and ananti-tumor cell proliferation assay.

ADCC ⁵¹ Chromium Release Assay

The ⁵¹ chromium release assay is a measure of effector cell cytotoxicactivity. For the ADCC ⁵¹ chromium release assay, we used a modificationof the method described by R. Perlmann and G. Perlmann, "ContactualLysis Of Antibody Coated Chicken Erythrocytes By Purified Lymphocytes",Cell Immunol., 1, p. 300 (1970).

We labelled target cells with ⁵¹ Cr (sodium chromate) DuPont - NewEngland Nuclear, catalog # NEZ-030S; 1mCi/ml concentration! as follows.2.5×10⁶ or 5.0×10⁶ target cells were pipetted into a 17×100 mm plasticcentrifuge tube and incubated with, respectively, 0.05 ml ⁵¹ Cr or 0.1ml ⁵¹ Cr for 75 minutes at 37° in a 5% CO₂ humidified atmosphere (FormaScientific Water Jacketed Gas Processor Incubator - Model 3315, Forma,Marietta, Ohio). The tube remained loosely capped and was shaken oncemidway during the 75 minute incubation period. To wash the cells, weadded 5 ml of a wash medium containing RPMI-1640 (Gibco Laboratories,Grand Island, N.Y.) supplemented with heat inactivated 5% fetal calfserum (Gibco Laboratories) and spun the tube in a Beckman TJ-6centrifuge equipped with a TH-4 rotor (Beckman Instruments, Palo Alto,Calif.) at 1200 rpm for 5 min. We removed the supernatant and added 5.0ml (for 2.5×10⁶ labelled cells) or 10.0 ml (for 5.0×10⁶ labelled cells)wash medium to give a final concentration of 5.0×10⁵ labelled cells/ml.We then apportioned 1.0 ml of the labelled cells into 4 ml tubes andgrouped them for treatment as follows.

The first group of target cells, which were not to be labelled withantibody, were washed 2 times with 1.5 ml wash medium and spun down for5 min. at 1200 rpm. The supernatant was removed and the target cellswere then resuspended in 1.0 ml wash medium to a concentration of 5×10⁵viable cells/ml. We then adjusted the cells to a final concentration of5×10⁴ viable cells/ml in wash medium.

The second group of target cells, which were to be labelled withantibody, were spun for 5 minutes at 1200 rpm. We removed thesupernatant and resuspended the pelletized target cells in 0.10 to 0.25ml of the appropriate antibody. The target cell/antibody mixture wasincubated for 30 minutes, with shaking, at room temperature. We thenadded 1.5 ml wash medium to the mixture and spun the cells for 5 minutesat 1200 rpm. We removed the supernatant and repeated the washing stepthree times. We then resuspended the target cells in wash medium toprovide a final concentration of 5×10⁴ viable cells/ml.

We prepared the effector cells (activated by the processes of thisinvention or unactivated) as follows. We washed the effector cells in 10ml of a medium containing RPMI-1640 and spun them down in a Beckman TJ-6centrifuge equipped with a TH-4 rotor for 5 min at 1200 rpm. We removedthe supernatant and resuspended the effector cells in wash medium to afinal concentration of 7×10⁶ viable effectors/ml for assays at aneffector:target ratio of 140:1 and 2.3×10⁶ viable effectors/ml forassays at an effector:target ratio of 47:1, i.e., so that 0.5 ml ofeffector cells were present for each target.

We plated the target cells and effector cells in a 96 well microtiterplate in triplicate (Costar Plastics, Cambridge, Mass.). Spontaneouswells were defined as those to which target cells and diluent were to beadded. Maximum wells were defined as those to which target cells anddetergent were to be added. Effector wells were those to which effectorcells, target cells and, in some cases, diluent were to be added, and inwhich effector cell:target cell ratios may also be in the range ofbetween about 140:1 and 1:1.

We pipetted 0.1 ml diluent, RPMI-1640 medium, into each spontaneous welland into each effector well having the highest effector:target ratios.No diluent was added to either the first three wells or the maximumwells of each target row. We then pipetted 0.15 ml of effector cellsinto the first three wells of each target row and made 1:3 serialdilutions of the effectors through the triplicates. To do this, weremoved 0.05 ml of effector cells from each of the first wells andtransferred them to each of the fourth wells using a pipette. We mixedthe fourth wells, removed 0.05 ml of effector cells from each of thefourth wells and added them to each of the seventh wells. We mixed eachof the seventh wells and removed 0.05 ml of their contents. We repeatedthis dilution procedure for each of two series of wells: 2, 5 and 8 and3, 6 and 9. We then added 0.1 ml of target cells (concentration of 5×10⁴viable cells/ml) to the wells of each target row. Subsequently, we added0.1 ml of detergent, Triton X-100, 1% in RPMI-1640 medium, to thosewells which contained target cells but no diluent, i.e., the maximumwells.

We then balanced the plates and spun them at 1000 rpm for 1 minute toeffect cell to cell contact. The plates were then incubated at 37° C.for 4 hours in a humidified atmosphere of 5% CO₂ in air. At the end of 4hours, we centrifuged the plates for 10 minutes at 2000 rpm. We thenremoved 0.1 ml of the supernatant from each well, while avoiding anypellet disturbance, and added it to a liquid scintillation counter minivial (Beckman Instruments). We then added 1 ml scintillation cocktail(Beckman HP/b) to each tube, capped the tube and shook it. We placedeach tube into a liquid scintillation counter (Beckman Instrumentsl,Model LS-3801) and began 1 minute counts to measure radioactivity.

The percent of specific target cell cytotoxicity was determined byaveraging triplicate count per minute ("CPM") measurements of ⁵¹chromium according to the following formula: ##EQU1##

Anti-Tumor Cell Proliferation Assay

The anti-tumor cell proliferation assay is a measure of the ability ofeffector cells to inhibit proliferation of tumor cells in vitro by ADCC.

Target tumor cells were collected from tissue culture flasks, washed inRPMI-1640 and resuspended in growth medium to achieve a concentration of1.25×10⁴ viable cells/ml. Wells 7-36 of a 96 well u-bottom sterilemicrotiter plate (Costar Plastics) were plated with 80 ml of culturegrowth medium using a Titer-Tek multichannel pipettor (made for FlowLaboratories by Eflab Oy, Finland).

Effector cells (activated by the processes of this invention orunactivated) were washed with RPMI-1640 and adjusted to a concentrationof 2.5×10⁶ viable cells/ml. We plated effector cells in replicate wellsof six to achieve six effector to target ratios: 200:1; 100:1; 50:1;25:1; 13:1; 6:1, in a total of 36 wells. Wells 1-6 were each plated with160 μl of effector cells from the 2.5×10⁶ concentration. We removed 80μl of effector cells from each of wells 7-12 and transferred them to,respectively, wells 13-18, and mixed them by repeat pipetting. This 1:2dilution was repeated out to well #36. We then discarded 80 μl fromwells 30-36. We added 20 μl of appropriate test antibody to each well ofthe experimental rows and 20 μl of culture medium to 36 control wells ofeffector cells ("Control No. 2"). Subsequently, we added 80 μl of targetcells to each well of the experimental rows (the 36 Control No. 2 wellsand the 36 control wells containing antibody only). Eighty μl of culturemedium were added to 36 control wells containing containing effectorsonly ("Control No. 1").

We incubated the plates at 37° C. in humidified 5% CO₂ and air for atotal time of 24 hours. After 20 hours of culture, 50 μl of ³H-thymidine (Dupont-New England Nuclear) from a concentration of 20μCi/ml was added to all wells. Fifteen minutes prior to harvesting allwells, we added 15 μl of trypsin (Gibco Laboratories, New York) 1:250concentration, to all wells to effect cell detachment from plastic. Allwells were subsequently harvested onto filter disks, PHD No. 240-1 GlassFiber Filter Strips (Cambridge Technology, Cambridge, Mass.) byautomated cell harvesting with a PHD cell harvester (CambridgeTechnology, Cambridge, Mass.). We placed dried filters into plasticmini-vials (Beckman Instruments) filled the vials with 2.0 ml ofscintillation cocktail (HP/b, Beckman), shook them vigorously, andcounted for beta in a liquid scintillation counter. Disintegrations perminute ("DPM") were calculated and six replicates were averaged ("x").Control wells of effectors and targets without antibody served as basefor tumor proliferation. Percent inhibition as a measure ofanti-proliferation was calculated according to the following formula:##EQU2##

When the anti-proliferation assay was conducted with "armed" effectorcells, we calculated percent inhibition as a measure ofantiproliferation according to the following formula: ##EQU3##

For the purposes of comparing the two assays, the ADCC activity ofeffector cells was evaluated in parallel by both the ⁵¹ chromium releaseassay and the anti-tumor cell proliferation assay. In these comparativeassays, we used human effector

                  TABLE I                                                         ______________________________________                                                Effector:                                                                     Target      % Specific                                                                              % Inhibition of                                 Assay # Ratio       cytotoxicity                                                                            proliferation                                   ______________________________________                                        1       100         51.7      107.4                                                   50          38.5      89.4                                                    25          30.4      59.2                                                    12.5        28.1      33.3                                                    6.25        18.4      25.0                                            2       100         73.7      99.7                                                    50          39.1      99.8                                                    25          29.6      91.9                                                    12.5        --        55.7                                                    6.25        --        34.9                                            3       100         61.5      84.2                                                    50          53.0      69.4                                                    25          41.2      46.1                                                    12.5        18.6      32.3                                                    6.5         6.7       24.0                                            ______________________________________                                    

⁵¹ Chromium release assay in vitro analyses have shown that ADCCsensitivity decreases in the presence of antibodies that induceantigenic modulation. Certain monoclonal antibodies (antigen associatedwith leukemia cells) have been shown to induce antigenic modulation suchas CALLA (J. Ritz et al., "Serotherapy of Acute Lymphoblastic Leukemiawith Monoclonal Antibodies", Blood, 58, pp. 141-52 (1981) and J. M.Pesando et al., "Distribution and Modulation of a HumanLeukemia-Associated Antigen (CALLA)", J. Immunology, 131, pp. 2038-45(1983)). As described below, the anti-proliferation assay was sensitiveenough to detect an ADCC mediated effect when directed toward stable andmodulating antigens. The assay was conducted in flasks undergoing eithera gentle rocking motion or no rocking motion. The rocking motionprovides cellular movement to facilitate cell to cell contact andpossibly minimize the effect of antigenic modulation. The assays wereperformed as described supra, except that rocking was accomplished byplacing the flasks on a rocker platform (Labquake Labindustries,Berkeley, Calif.) and set for approximately 40-50 rocking motions perminute. The tumor target cells were the Zbtu and Nalm-6 lines. Theantibodies used included GAGPA, RaNalm or anti-CALLA (# 7500,Becton-Dickinson, Mountain View, Calif.). The results of both theanti-proliferation assay and the ⁵¹ chromium release assay are shown inTable II.

                  TABLE II                                                        ______________________________________                                                    % Specific                                                                              % Inhibition                                            Target   Antibody cytotoxicity                                                                              rocked                                                                              not-rocked                                ______________________________________                                        Zbtu     none     6.1         0     0                                                  GAGPA    79.5        86.5  88.1                                      Nalm-6   none     3.4         0     0                                                  RaNalm   58.9        103.5 93.8                                               CALLA    7.1         47.3  10.9                                      ______________________________________                                    

Frozen Storage

We also employed these assays to evaluate the effect of frozen storage(-75° C.) on effector cell ADCC activity by ⁵¹ chromium release assays.The Zbtu tumor with GAGPA antibody was the target cell. Effector cellswere obtained from three healthy donors as described supra and frozenaccording to the technique in Pross et al., "The Standardization of NKCell Assays for Use in Studies of Biological Response Modifiers", J.Immunological Methods, 68, pp. 235-49 (1984). The cells were thawedafter 12-58 days and washed two additional times in standard wash mediumas described supra. The thawed effector cells were then activated asdescribed supra. Table III demonstrates cell viability before and afterfreezing, the ADCC activity as expressed by percent cytotoxicity andpercent recovery of viable cells after freezing.

                  TABLE III                                                       ______________________________________                                        % Viability     % Cytotoxicity                                                                              % Viable Cells                                  Donor Day 0    Day (X)  Day 0  Day (X)                                                                              Recovered                               ______________________________________                                        1     94       93 (12)  76     68 (12)                                                                              67                                      2     92       80 (34)  71     49 (34)                                                                              58                                      3     97       94 (58)  71     58 (58)                                                                              62                                      ______________________________________                                    

Tumor Treated

The animal tumor treated in vivo according to the processes of thisinvention in the following examples was Zbtu, a T-cell leukemia/lymphomaoriginated in C3Hf inbred mice by intrathymic injection of the Grosspassage A virus. Primary thymomas were excised, cells were suspended insterile saline and injected intraperitoneally into normal adult C3Hfrecipient mice to induce an ascites-form tumor. This tumor has beenserially transferred in vivo since 1970. It has an LD₁₀₀ of less than 10cells in C3Hf mice with an approximate mean survival time of 16 days. Asubline of this tumor has been adapted for growth in vitro as well withno apparent loss of tumorigenicity. Zbtu tumor cells express thefollowing surface antigens, detected by means of indirectimmunofluorescence, complement dependent serum toxicity, FACS(fluorescence activated cell sorter) analysis and ADCC:

(1) Thy 1.2

(2) gP70

(3) p12

(4) Gross passage A virus

Antibodies used in the in vivo treatment of mice according to theprocesses of this invention were GAGPA antibodies.

Example 1

C3Hf strain inbred mice (bred at University of Minnesota Mouse Colonyand available from Microbiological Associates, Walkersville, Md.) werealloprimed three times, on days 0, 7, and 14, by transplanting a 5×5 mmsection of whole spleen tissue from a C57Bl/6J (Jackson Laboratories,Bar Harbor, Me.) strain donor subcutaneously into the ear capsule. Onday 17, host spleens were aseptically excised from both the alloprimedmice and normal age matched controls. Spleen cells were harvested intoComplete Dulbecco's Minimal Essential Medium (Gibco Laboratories, GrandIsland, N.Y.) supplemented with 5% fetal calf serum, by passage througha stainless mesh screen, type 304, no. cx-30 (Small Parts Inc., Miami,Fla.). The spleen cells were cultured in one-way mixed lymphocytecultures ("MLC") J. C. Cerottini et al., "Generation Of CytotoxicT-Lymphocytes In Vitro I. Response Of Normal And Immune Mouse SpleenCells In Mixed Leukocyte Culture", J. Exp. Med., 140, p. 703 (1974)! for3 to 7 days with C57Bl/6J stimulator cells at a concentration of 1.0×10⁷cells/ml for both responders and stimulators at a 1:1 ratio. Respondercells were used as effector cells in a ⁵¹ chromium release assay (asdescribed supra) against GAGPA antibody labelled Zbtu tumor targets(Table IV).

Responder cells were also used as effector cells for in vivo treatmentof mice bearing greater than 1×10⁵ Zbtu leukemia cells. A balancedsaline solution containing 1.0×10⁷ effector cells and, in some cases,GAGPA antibody, was administered intravenously to leukemic C3Hf mice(Table V). In Table VI, the Group A mice represented 10 mice, which hadeach received an intravenous injection of 0.1 ml GAGPA and 0.2 mlbalanced saline solution. Group B mice represented 10 mice, which hadeach received an intravenous injection of 0.1 ml normal goat serum and0.2 ml (1.0×10⁷ viable cells) effector cells in balanced salinesolution. Group C mice represented 10 mice, which had each received anintravenous injection of 0.1 ml GAGPA and 0.2 ml (1.0×10⁷) effectorcells in balanced saline solution.

                  TABLE IV                                                        ______________________________________                                        Percent Specific ADCC - Day 5 MLC                                             Effector:Target Ratio                                                                           Unprimed Primed                                             ______________________________________                                        140:1             19.9     56.0                                               47:1              10.4     47.0                                               16:1              8.3      31.2                                               ______________________________________                                    

                  TABLE V                                                         ______________________________________                                        Percent Survival of Treated Tumor-Bearing Mice                                Group    6 weeks      10 weeks 18 weeks                                       ______________________________________                                        A        20           0        0                                              B        10           0        0                                              C        100          90       40                                             ______________________________________                                    

Example 2

We isolated effector cells from peripheral blood of healthy human donorsas follows. One unit (450 cc) of whole peripheral blood was drawn from adonor by venipuncture into a CPDA blood bag containing 63 mlanticoagulant-citrate phosphate-dextrose adenine-1 solution and placedin either ten 50 ml tubes. The blood sample was then centrifuged in aBeckman TJ-6 centrifuge equipped with a TH-4 rotor for 10 min at 1200rpm. As a result of the centrifugation, the red cells pelletized and theleukocytes formed a buffy coat which was harvested by pipetting throughthe top layer of plasma.

We collected the buffy coats from each tube and pooled them in a 50 mltube before density centrifugation to further sort out effector cells.We then added Hank's balanced salt solution to a volume of 17.3 ml andcarefully layered the volume onto 14 ml Ficoll-hypaque in a 50 ml glasstube. The tube was centrifuged in a Beckman TJ-6 centrifuge equippedwith a TH-4 rotor for 30 min at 1400 rpm and at a temperature of 20° C.We removed and discarded the top layer using a pipette and collected theinterface into a 50 ml glass tube and added 30 ml sterile wash medium(RPMI-1640 supplemented with at least 2% autologous serum). The tube wascentrifuged for 15 min at 1400 rpm. We removed the supernatant andresuspended the effector cells in 8-10 ml wash medium. We then removed asmall amount of the suspension to a microtube to count 1:100.Subsequently, we removed desired volumes of effector cells and washedthem twice in wash medium or experimental medium. The leukocytes weresuspended in RPMI 1640 culture medium at a concentration of 1×10⁷cells/ml.

We then cultured the leukocytes in a one-way mixed lymphocyte culture,prepared according to the method described supra, in a 1:1 ratio witheither allogeneic (pooled from non-related donors) or xenogeneic (pooledfrom C57B1/6J inbred mouse spleens) stimulator cells which had beenirradiated to 2500R. Responder cells were used as effector cells in a ⁵¹chromium release ADCC assay against unlabelled or GAGPAantibody-labelled Zbtu tumor targets before being mixed with thestimulator cells (day 0) and after remaining in culture with thestimulator cells for between 3 and 7 days. Table VI indicates theincrease in ADCC activity, as measured in a ⁵¹ chromium release assay(effector:target ratio=140:1) of effector cells treated with the mixedlymphocyte culture.

                  TABLE VI                                                        ______________________________________                                        % Specific ADCC                                                               Stimulator Day 0   Day 3      Day 5 Day 7                                     ______________________________________                                        Allogeneic 38.1    61.5       86.2  79.5                                      Xenogeneic 38.1    53.2       66.6  59.7                                      ______________________________________                                    

Example 3

We obtained 450 ml peripheral blood from healthy human donors accordingto the procedure set forth in Example 2, except that the blood wasplaced into two 250 ml bottles instead of ten 50 ml tubes and theleukocyte-containing buffy coats were not pooled. We resuspended theleukocytes in RPMI-1640 culture medium, containing Gentamicin (5 μg/ml),at a concentration of 1×10⁷ cells/ml in the presence of varying amountsof gamma interferon (Cellular Products, Buffalo, N.Y.). The gammainterferon was present in amounts ranging between 100 and 1000 units perml. The cultures obtained were maintained for between 1 and 4 days ateither 4° C. in air or 37° C. in a 5% CO₂ atmosphere incubator. Thetreated effector cells were used in a ⁵¹ chromium release ADCC assayagainst unlabelled or GAGPA antibody Zbtu labelled tumor targets beforebeing mixed with the interferon and after each interferon treatment overthe 1-4 day treatment period. Table VII indicates the increase in ADCCactivity of the treated effector cells, as measured in the ⁵¹ chromiumrelease assay (effector:target ratio=140:1).

                  TABLE VII                                                       ______________________________________                                        % Specific ADCC                                                               Units        4° C.    37° C.                                    IFN  Day 0   Day 1   Day 2 Day 4 Day 1 Day 2 Day 4                            ______________________________________                                        100  36.3    39.1    43.7  45.9  42.8  46.6  39.1                             200  36.3    ND      ND    ND    49.7  51.9  59.3                             600  36.3    43.8    51.1  49.6  36.4  53.6  51.2                             1000 36.3    33.2    36.1  39.4  ND    ND    ND                               ______________________________________                                         % specific ADCC are mean values                                               ND = Not determined.                                                     

Example 4

We isolated human leukocyte effector cells and suspended them inRPMI-1640 culture medium, containing Gentamicin (5 μg/ml), at aconcentration of 1×10⁷ cells/ml as in Example 3. We added recombinantinterleukin-2 (Genzyme Corporation, Boston, Mass.) to effector cellcultures in amounts ranging between 30 and 750 units per ml.

The cultures thus obtained were maintained for between 1 and 4 days ateither 4° C. in air or 37° C. in a 5% CO₂ atmosphere incubator. Thetreated effector cells were used in a ⁵¹ chromium release ADCC assayagainst unlabelled or GAGPA antibody labelled Zbtu tumor targets bothbefore being mixed with the interleukin-2 and after each interleukin-2treatment over the 1-4 day treatment period. Table VIII indicates theincrease in ADCC activity of the treated effector cells, as measured inthe ⁵¹ chromium release assay (effector:target ratio=140:1).

                  TABLE VIII                                                      ______________________________________                                        % Specific ADCC                                                               Units        4° C.    37° C.                                    IL-2 Day 0   Day 1   Day 2 Day 4 Day 1 Day 2 Day 4                            ______________________________________                                        30   43.2    36.9    46.3  51.2  51.6  62.3  55.4                             50   43.2    ND      ND    ND    39.5  51.9  60.7                             100  43.2    48.7    49.6  54.6  ND    ND    ND                               250  43.2    39.2    55.4  49.3  56.4  62.1  57.8                             500  43.2    ND      ND    ND    60.6  63.7  59.4                             750  43.2    36.1    52.7  61.1  61.4  60.9  58.3                             ______________________________________                                         % specific ADCC are mean values                                               ND = Not determined.                                                     

Example 5

Human leukocyte effector cells were isolated and suspended in RPMI-1640culture medium, containing Gentamicin (5 μg/ml), at a concentration of1×10⁷ cells/ml as in Example 4. We then added gamma interferon (CellularProducts, Buffalo, N.Y.) to the cultures in amounts varying between100-1000 units/ml and maintained the cultures for between 1 and 4 daysat 37° C. in a 5% CO₂ atmosphere incubator. The effector cells were thenwashed three times in RPMI-1640 culture medium to remove the addedinterferon. We then added interleukin-2 (Genzyme Corporation, Boston,Mass.) to the cells in amounts varying between 100-600 units/ml, andmaintained the cells for between 1 and 4 days at 37° C. in a 5% CO₂atmosphere incubator. The effector cells were then used in a ⁵¹ chromiumrelease ADCC assay against unlabelled targets of Zbtu tumors and ofNalm-6 tumors, GAGPA antibody-labelled Zbtu tumor targets and rabbitanti-Nalm-6 labelled Nalm-6 tumor targets before being mixed with thelymphokines and after each lymphokine treatment over the treatmentperiod and the percent ADCC activity was determined.

Table IX depicts the increase in ADCC activity of the treated effectorcells (effector:target ratio=140:1).

                  TABLE IX                                                        ______________________________________                                        % Specific ADCC                                                                                      Rabbit anti-Nalm-6                                                            labelled Nalm-6                                        GAGPA-labelled Zbtu targets                                                                          targets                                                Group  Day 0   Day 2   Day 4 Day 6 Day 0  Day 4                               ______________________________________                                        A      41.4    66.7    89.1  76.3  36.3   75.2                                B      41.4    ND      79.8  73.6  36.3   88.7                                C      41.4    90.1    106.5 101.3 36.3   94.3                                D      41.4    88.2    91.4  ND    36.3   ND                                  E      41.4    75.5    86.3  89.1  36.3   ND                                  ______________________________________                                         A  600 units IFN, 500 units IL2                                               B  600 units IFN, 250 units IL2                                               C  200 units IFN, 600 units IL2                                               D  600 units IFN, 600 units IL2                                               E  1000 units IFN, 100 units IL2                                         

For all groups, effector cells were cultured for equal time duration inIFN (first) and IL-2 (second). ND - Not determined.

Example 6

We isolated human leukocyte effector cells as in Example 5 and suspendedthem in RPMI-1640 with 5% autologous serum at a concentration of 1×10⁷cells/ml. We then measured % ADCC of these cells in a ⁵¹ chromiumrelease ADCC assay.

Subsequently, we washed the effector cells three times in RPMI-1640medium to remove the autologous serum and suspended them in RPMI 1640culture medium, containing Gentamicin (5 μg/ml), and which wassupplemented with either 1% Ultroser G, 5% autologous serum, 5% fetalcalf serum or 5% pooled allogenic serum. We maintained the cultures forbetween 1 and 4 days at either 4° C. in air or 37° C. in a 5% CO₂atmosphere. The effector cells were used in a ⁵¹ chromium release ADCCassay against unlabelled targets of Zbtu tumors and of Nalm-6 tumors,GAGPA antibody labelled Zbtu tumor targets, Anti-CALLA labelled Nalm-6targets and rabbit anti-Nalm-6 labelled Nalm-6 tumors, before beingmixed with any RPMI-1640 supplemented medium and after each treatmentperiod and the percent ADCC activity was determined.

Table X depicts the increase in ADCC activity of the treated effectorcells (effector:target ratio=140:1) against GAGPA antibody labelled Zbtutumor targets. Table XI depicts the increase in ADCC activity of treatedeffector cells (effector:target ratio=140:1) against Nalm-6 tumortargets labelled with either Anti-CALLA or rabbit anti-Nalm-6antibodies.

                  TABLE X                                                         ______________________________________                                        % Specific ADCC                                                                       4° C.  37° C.                                           Culture                                                                             Day     Day     Day   Day   Day   Day   Day                             Medium                                                                              0       1       2     4     1     2     4                               ______________________________________                                        A     38.6    39.6    41.3  38.6  39.3  40.2  42.8                            B     38.6    43.1    36.3  31.8  41.4  33.2  36.8                            C     38.6    68.7    83.2  79.4  81.5  93.1  86.4                            D     38.6    64.4    69.7  91.5  86.7  78.5  85.3                            ______________________________________                                    

                  TABLE XI                                                        ______________________________________                                        % Specific ADCC                                                                      Anti-CALLA        Rabbit anti-Nalm-6                                          labelled Nalm-6   labelled Nalm-6                                      Culture  targets (37° C.)                                                                           targets (37° C.)                          Medium   Day 0   Day 2       Day 0 Day 2                                      ______________________________________                                        A        12.1    17.3        32.1  31.1                                       B        12.1    13.4        32.1  39.5                                       C        12.1    37.8        32.1  69.4                                       D        12.1    39.1        32.1  61.6                                       ______________________________________                                         A  RPMI1640 supplemented with 1% Ultroser G (LBK Industries, Gaithersburg     Maryland)                                                                     B  RPMI1640 supplemented with 5% autologous serum                             C  RPMI1640 supplemented with heat inactivated 5% fetal calf serum (Gibco     Laboratories, Grand Island, New York)                                         D  RPMI1640 supplemented with 5% pooled allogenic serum                  

Example 7

We isolated human effector cells as described in Example 6 and suspendedthem in either RPMI-1640, containing Gentamycin (5 μg/ml), andsupplemented with 5% autologous serum or 1% Ultroser G (non-activatedcells) or RPMI-1640 supplemented with 5% fetal calf serum (activatedcells), to attain a cell concentration in each suspension of 1×10⁷cells/ml. We measured the percent anti-proliferation activity by ADCC ofthe treated effector cells against Zbtu target cells, labelled withGAGPA antibodies and Nalm-6 target cells, labelled with rabbitanti-Nalm-6 antibodies after a 1 or 2 day treatment period.

Table XII demonstrates the inhibition of proliferation of target tumorcells by the treated effector cells.

                  TABLE XII                                                       ______________________________________                                        % Anti-Proliferation                                                                    Zbtu targets                                                                              Nalm-6 targets                                          Effector    Day 1   Day 2     Day 1 Day 2                                     ______________________________________                                        Non-activated                                                                             24.3    32.1      20.1  19.6                                      Activated   84.2    78.8      82.6  92.3                                      ______________________________________                                    

The use of monoclonal antibodies to attach and thereby "arm" an effectorcell was evaluated in both the ⁵¹ chromium release assay and theanti-proliferation assay. Monoclonal antibodies capable of recognizingantigens expressed on the Zbtu cell surface were analyzed for theirability to mediate ADCC. The IgG2a class monoclonal antibody anti-Thy1.2 (#630021, Miles Scientific) and the IgG2b class monoclonal antibodyanti-Thy 1.2 (#1330, Becton-Dickinson, Mountain View, Calif.) recognizethe Thy 1.2 antigen expressed on murine "T" cells and the Zbtu tumorcell line. The assays used Zbtu target cells unlabelled or labelled withGAGPA as a control. The human effector cells were either "unarmed" (notmanipulated) or armed with antibody (GAGPA or Thy 1.2). The results ofthese assays are shown in Table XIII.

                  TABLE XIII                                                      ______________________________________                                        Target                     % Specific                                                                             % Inhibition                              cell   Antibody   Effector cytotoxicity                                                                           proliferation                             ______________________________________                                        Zbtu   None       unarmed  5.9      0                                         Zbtu   GAGPA      unarmed  79.1     86.6                                      Zbtu   GAGPA      armed    65.3     79.7                                      Zbtu   THY 1.2(2a)                                                                              unarmed  78.6     104.5                                     Zbtu   THY 1.2(2a)                                                                              armed    74.3     88.8                                      Zbtu   THY 1.2(2b)                                                                              unarmed  82.5     86.3                                      Zbtu   THY 1.2(2b)                                                                              armed    75.4     89.8                                      ______________________________________                                    

Example 9

We obtained leukocytes from the peripheral blood of each of eight humandonors diagnosed with leukemia by drawing 50 ml of blood into a 60 mlsyringe containing 2 ml of heparin (heparin sodium injection-1000 USPunits/ml, Elkins-Sinn, Inc., Cherry Hill, N.J.) anticoagulant. Theleukocytes were subsequently isolated as described in Example 2.Effector cells were analyzed by ⁵¹ chromium release ADCC assay againstunlabelled and GAGPA antibody-labelled Zbtu tumor targets and autologoustumor targets without further manipulation as controls.

Cultured effector cells were stimulated with 10% allogeneic or 10%xenogeneic serum, containing Gentamycin (5 μg/ml), at a concentration of1×10⁷ cells/ml for 1 to 5 days and re-assayed for ADCC. Table XIVdemonstrates the ability of leukemia donors' effector cells to mediateADCC against various Zbtu tumor target cells and autologous tumor targetcells.

                  TABLE XIV                                                       ______________________________________                                        % Specific ADCC                                                                                  Zbtu    Autologous                                                            targets tumor targets                                             Diag-    Treat-         Day  Day  Day  Day                             Patient #                                                                            nosis    ment    Status 0    2    0    2                               ______________________________________                                        1      ALL      CC      Acute  32.6 58.4 31.1 53.4                            2      CML      CC      Relapse                                                                              14.0 21.1 13.3 37.7                            3      CLL      CC      Rem    49.5 83.4 ND   ND                              4      ALL      CC      Rem    19.8 62.7 ND   ND                              5      CLL      CC      Relapse                                                                              8.7  12.2 5.4  18.7                            6      CML      BMT     Rem    31.4 60.9 ND   ND                              7      T lymph  CC      Rem    23.2 71.1 ND   ND                              8      CLL      CC      Rem    45.1 52.4 ND   ND                              ______________________________________                                         Definitions:                                                                  ALL  acute lymphocytic leukemia                                               CLL  chronic lymphocytic leukemia                                             CML  chronic myelogenous leukemia                                             T lymph  "T" cell lymphoma                                                    CC  combined chemotherapy                                                     BMT  bone marrow transplant (allogeneic)                                      Rem  remission                                                                ND  not determined.                                                      

Patients 2 and 5 in Table XIV had extensive contamination of tumor cellsin the peripheral blood specimen. In both patients, greater than 80% ofthe effector cells were malignant. Therefore, the adjusted concentrationof effector cells in each of these patient's blood samples wasequivalent to approximately 20% of recovery of the concentration ofeffector cells in blood samples obtained from the other patients. Inaddition, patients 3, 4, 6, 7 and 8, who were in remission at the timeof treatment, did not have any circulating tumor cells in theirperipheral blood.

Example 10

We obtained leukocytes from the peripheral blood of each of four humanpatients diagnosed with leukemia. Blood was drawn and leukocytesisolated as described in Example 8. The leukocytes were cultured for 24hours at 37° C. in a humidified CO₂ (10%) incubator in RPMI-1640supplemented with 10% fetal calf serum. The effector cells were analyzedby ⁵¹ chromium release ADCC assay against the Zbtu tumor target cellsunlabelled or labelled with either GAGPA polyclonal antibody, Thy 1.2-2aor Thy 1.2-2b monoclonal antibodies. The results are listed in Table XV.

                  TABLE XV                                                        ______________________________________                                        Patient                                                                             Diag-   Treat             Antibody                                                                             THY 1.2:                               #     nosis   ment    Status                                                                              None  GAGPA  (2a) (2b)                            ______________________________________                                        1     CML     CC      Rem   0     16     39   13                              2     ALL     CC      Rem   0     68     71   64                              3     CML     None    Chronic                                                                             1     22     45   16                              4     CML     None    Chronic                                                                             1     39     65   23                              ______________________________________                                    

Example 11

We obtained leukocytes from the peripheral blood of each of five humanpatients diagnosed with leukemia. The blood was drawn and leukocytesisolated as described in Example 8. The effector cells were treated byone of five procedures: (1) unmanipulated; (2) cultured for 48 hours inRPMI-1640 supplemented with 10% fetal calf serum (3) cultured for 48hours in RPMI-1640 supplemented with IL-2 (70 units/ml); (4) culturedfor 48 hours in RPMI-1640 supplemented with IFN (300 units/ml); (5)cultured sequentially, first for 24 hours in RPMI-1640 supplemented withIFN (300 units/ml), washed two times in standard wash medium (asdescribed supra), and cultured for an additional 24 hours in IL-2 (70units/ml). All cultures were incubated in a humidified CO₂ (10%)incubator at 37° C. The effector cells were analyzed by ⁵¹ chromiumrelease ADCC assay against Zbtu tumor target cells labelled with GAGPApolyclonal antibody. The results are listed in Table XVI.

                  TABLE XVI                                                       ______________________________________                                        Patient                                                                             Diag-   Treat-        Day                 IFN/                          #     nosis   ment    Status                                                                              0    FCS  IL-2 IFN  IL-2                          ______________________________________                                        1     ALL     None    Acute 23   --   --   30   31                            2     CML     None    Chronic                                                                             39   45   45   --   --                            3     CML     CC      Rem   16   40   28   --   --                            4     CML     CC      Relapse                                                                             12   33   20   --   21                            5     CML     None    Chronic                                                                             22   51   --   --   --                            ______________________________________                                    

Example 12

We analyzed effector cells which had been activated according to theprocess of this invention by flow cytometry for binding of monoclonalantibodies Leu-11a by direct immunofluorescence and Leu-11b by indirectimmunofluorescence. Leu-11a and Leu-11b are antibodies which arebelieved to recognize the Fc receptor on large granular lymphocytes andgranulocytes (B. Perussia et al., "The Fc Receptor For IgG On HumanNatural Killer Cells: Phenotypic, Functional And Comparative StudiesUsing Monoclonal Antibodies", J. Immunol., 133, p. 180 (1984)).

We isolated human leukocyte effector cells as in Example 5 and treatedthe cells with either autologous serum or fetal calf serum, γ-IFN, IL-2or a γ-IFN+IL-2 mixture according to the procedures set forth in,respectively, examples 7, 3, 4 and 5. The specific times andtemperatures of treatment, as well as the concentrations of serum orlymphokine used are set forth in Table XVII.

We took cells from each treatment group and suspended them in phosphatebuffered saline (PBS, 0.01M phosphate, 0.15M NaCl, pH 7.2-7.4) at aconcentration of 1×10⁶ viable cells/ml and washed the cells bycentrifugation in a Beckman TJ-6 centrifuge equipped with a TH-4 rotorfor 5 min at 1200 rpm. We divided the cell pellets into four groups andresuspended each group in one of the following:

Group A: Leu-11a, 5 μl antibody, 40 μl PBS, to achieve a 1:9 dilution

Group B: Mouse IgGl, 6 μl of 200 μg/ml concentration for 1×10⁶, cellsnon-specific control!

Group C: Leu-11b, 5 μl antibody, 40 μl PBS, to achieve a 1:9 dilution

Group D: MOPC 104E purified mouse myeloma protein, 5 μl of a 1:5dilution per 1×10⁶ cells non-specific control!.

Each of the four groups of cells were incubated for 30 min at 4° C. in acontainer which was covered from ambient light. We then washed theeffector cells twice, each time adding 1.0 ml PBS and centrifuging themixture for 5 min at 1200 rpm.

The cells of each of Group A and Group B were then resuspended in 1.0 mlPBS to a concentration of 1.0×10⁶ viable cells/ml and analyzed.

The cells of each of Group C and Group D were resuspended in affinitypurified fluorescein isothiocyanate (FITC) conjugated goat anti-mouseIgM antibody (Tago, Inc., Burlingame, Calif.) by adding 0.10 ml of a1:50 antibody dilution in PBS. These effector cells were then incubatedfor an additional 30 min at 4° C. in a container covered from the light.We washed each group of cells twice, each time adding 1.0 ml PBS andcentrifuging the mixture for 5 min at 1200 rpm. The cells of each groupwere then re-suspended in 1.0 ml PBS to a concentration of 1.0×10⁶viable cells/ml and analyzed.

The effector cells were then evaluated for immunofluorescence by flowcytometry analysis in a FACS IV cell sorter both before being treatedwith the serum or lymphokines and after serum or lymphokine treatmentand exposure to the antibodies. FITC was excited by using 400 mW of488nM light (argon laser). Fluorescence emission was collected by a 90°angle focusing lens. Green light passed through a 530 nM band passfilter into a photomultiplier tube (EMI no. 99224A). Green fluorescence90° angle light scatter peak amplitudes of electrical signals wereconverted from analog to digital, amplified by a linear amplifier andassigned into 256 channels. Histograms were plotted with thefluorescence channels on the X-axis and the relative number of positivecells on the Y-axis. Non-specific control values (Groups B and D) weresubtracted from the experimental values to obtain mean channel valuesand % positive cells (Mean Channel Experimental-Mean ChannelControl=Mean Channel Difference, and % positive cells). The % positivecell data reflects the number of cells which specifically boundantibody. The mean channel data indicates the intensity ofimmunofluorescence on the positive cells and, thus, the relativequantity of antibody binding which occurred.

The effector cells were also used in a ⁵¹ chromium release ADCC assay(effector:target ratio=140:1) against GAGPA antibody-labelled Zbtu tumortargets both before being treated with the serum or lymphokines andafter those treatments and exposure to the antibodies.

                                      TABLE XVII                                  __________________________________________________________________________             Day 0          Day 1          Day 2                                           Mean           Mean           Mean                                            Channel                                                                            % Positive                                                                              Channel                                                                            % Positive                                                                              Channel                                                                            % Positive                        Effector Difference                                                                         Cells                                                                              % ADCC                                                                             Difference                                                                         Cells                                                                              % ADCC                                                                             Difference                                                                         Cells                                                                              % ADCC                       __________________________________________________________________________    Control (4° C.)                                                                 62.2 16.9 26.4 64.2 18.1 22.8 60.6 17.2 ND                           Control (37° C.)                                                                58.6 18.1 31.4 52.9 21.9 27.5 56.9 20.7 30.7                         FCS (37° C.)                                                                    ND   ND   ND   77.6 34.9 59.8 91.1 31.6 56.3                         IFN (37° C.)                                                                    ND   ND   ND   76.8 24.2 49.1 77.2 26.3 ND                           IL-2 (37° C.)                                                                   ND   ND   ND   88.2 20.4 52.6 110.2                                                                              24.4 61.5                         IFN + IL-2 (37° C.)                                                             ND   ND   ND   85.0 37.7 61.4 73.8 34.8 60.9                         __________________________________________________________________________     Control  RPMI1640 supplemented with 5% autologous serum                       FCS  RPMI1640 supplemented with 5% heat inactivated fetal calf serum          IFN  RPMI1640 containing 600 units IFN/10.sup.6 cells                         IL2  RPMI1640 containing 500 units IL2/10.sup.6 cells                         IFN + IL2  RPMI1640 containing 200 units IFN + 600 units IL2/10.sup.6         cells                                                                         ND -- Not determined                                                     

Having described our invention with particular reference to thepreferred form thereof, it will be apparent to those skilled in the artto which the invention pertains that various changes and modificationsmay be made therein without departing from the spirit and scope of theinvention as defined by the claims appended hereto.

We claim:
 1. A process for increasing the antibody dependentcell-mediated cytotoxicity (ADCC) of leukocyte effector cells thatexpress or have the potential to express Fc receptors for antibodycomprising:a) activating the effector cells in vitro by combining themwith interleukin-2 for a sufficient period of time to increase the ADCCactivity of said effector cells to at least 1.25 to 125 times that ofeffector cells that have not been activated; and b) contacting theactivated effector cells with a target cell that is susceptible to ADCCin the presence of an antibody which is capable of ADCC mediation andwhich primarily immunoreacts with an antigen that is specific to thetarget cell or that is present in higher densities on the target cellthan on normal cells.
 2. A process for increasing the antibody dependentcell-mediated cytotoxicity (ADCC) of leukocyte effector cellscomprising:a) selectively isolating leukocytes that express or have thepotential to express Fc receptors for antibody; b) activating theeffector cells in vitro by combining them with interleukin-2 for asufficient period of time to increase the ADCC activity of said effectorcells to at least 1.25 to 125 times that of cells that have not beenactivated; and c) contacting the activated effector cells with a targetcell that is susceptible to ADCC in the presence of an antibody which iscapable of ADCC mediation and which primarily immunoreacts with anantigen that is specific to the target cell or that is present in higherdensities on the target cell than on normal cells.
 3. A method ofobtaining and using leukocyte effector cells that express or have thepotential to express Fc receptors for antibody in effective target cellkilling quantities comprising:a) selectively isolating leukocytes thatexpress or have the potential to express Fc receptors for antibody; b)activating the effector cells in vitro by combining them withinterleukin-2 for a sufficient period of time to increase the ADCCactivity of said effector cells to at least 1.25 to 125 times that ofeffector cells that have not been activated; c) proliferating theactivated effector cells using in vitro tissue culture reproductionmethods; and d) contacting the proliferating effector cells with ADCCsusceptible target cells in the presence of an antibody which is capableof ADCC mediation and which primarily immunoreacts with an antigen thatis specific to the target cell or that is present in higher densities onthe target cell than on normal cells.
 4. A method of obtaining leukocyteeffector cells that express or have the potential to express Fcreceptors for antibody in effective target cell killing quantitiescomprising:a) selectively isolating leukocytes that express or have thepotential to express Fc receptors for antibody; b) proliferating theeffector cells using in vitro tissue culture reproduction methods; and(c) activating the proliferated effector cells in vitro by combiningthem with interleukin-2 for a sufficient period of time to increase theADCC activity of said effector cells to at least 1.25 to 125 times thatof effector cells that have not been activated.
 5. A method forpreserving, reviving, and using ADCC mediating effector cellscomprising:a) obtaining an effective target cell killing quantity ofleukocyte effector cells that express or have the potential to expressFc receptors for antibody; b) activating the effector cells in vitro bycombining them with interleukin-2 for a sufficient period of time toincrease the ADCC activity of said effector cells to at least 1.25 to125 times that of effector cells that have not been activated; c)freezing the effector cells to store them in indefinite periods of time;d) thawing the effector cells; and e) contacting the activated effectorcells with ADCC susceptible target cells in the presence of an antibodywhich is capable of ADCC mediation and which primarily immunoreacts withan antigen that is specific to the target cell or that is present on thetarget cell in higher densities than on normal cells.
 6. A method forpreserving, reviving, and using ADCC mediating effector cellscomprising:a) obtaining an effective target cell killing quantity ofleukocyte effector cells that express or have the potential to expressFc receptors for antibody; b) freezing the effector cells to store themfor indefinite periods of time; c) thawing the effector cells; d)activating the effector cells in vitro by combining them withinterleukin-2 for a sufficient period of time to increase the ADCCactivity of said effector cells to at least 1.25 to 125 times that ofeffector cells that have not been activated; and e) contacting theactivated effector cells with ADCC susceptible target cells in thepresence of an antibody which is capable of ADCC mediation and whichprimarily immunoreacts with an antigen that is specific to the targetcell or that is present on the target cell in higher densities than onnormal cells.
 7. A process for increasing the antibody dependentcell-mediated cytotoxicity (ADCC) of leukocyte effector cells thatexpress or have the potential to express Fc receptors for antibodycomprising:a) activating the effector cells in vitro by combining themwith interleukin-2 for a sufficient period of time to increase the ADCCactivity of said effector cells to at least 1.25 to 125 times that ofeffector cells that have not been activated; and b) "arming" theactivated effector cells in vitro with an antibody which is capable ofADCC mediation and which primarily immunoreacts with an antigen that isspecific to a target cell that is susceptible to ADCC or that is presentin higher densities on said target cell than on normal cells.
 8. Theprocess of claim 7, further comprising contacting the armed effectorcells with the target cell which is susceptible to ADCC.
 9. The processof claim 8, further comprising contacting the armed activated effectorcells with the target cell in the presence of:a) an additional amount ofthe antibody used for arming; or b) a second antibody which is capableof ADCC mediation and which recognizes a second antigen epitope that isspecific to the target cell or that is present in higher densities onthe target cell than on normal cells.
 10. An effector cell activated byinterleukin-2 and armed with an antibody according to the process ofclaim
 7. 11. The process of claim 2, 3, 4, 5, 6, 7, 8 or 9 wherein theeffector cells are selectively isolated by:a) obtaining cells having adiameter in the range from 7 μm to 25 μm; or b) labelling cells withmonoclonal antibody and separating by positive selection.
 12. Theprocess of claim 1, 2, 3, 5, 6, 7, 8, or 9 wherein the ADCC mediatingantibody is monoclonal.
 13. The process of claim 1, 2, 3, 5, or 6,wherein the effector cells are armed with the ADCC mediating antibodyprior to contacting with said target cells.
 14. The process of claim 1,2, 3, 5, or 6 further comprising contacting the activated effector cellswith target cells in the presence of a second antibody which ismonoclonal and which primarily immunoreacts with an antigen that isspecific to the target cell or that is present in higher densities onthe target cell than on normal cells.
 15. A composition for use in thekilling of disease target cells consisting essentially of:a) a quantityof leukocyte effector cells that express Fc receptors for antibody, andthat have been activated by combining them in vitro with interleukin-2for a sufficient period of time to increase the ADCC activity of saideffector cells to at least 1.25 to 125 times that of effector cells thathave not been activated; and b) a quantity of interleukin-2 sufficientfor such activation; wherein said activated effector cells are capableof mediating ADCC against disease target cells in the presence of anantibody which primarily immunoreacts with an antigen that is specificto the disease target cell or that is present in higher densities on thedisease target cell than on normal cells, so that such composition hascytotoxic effect specifically on disease target cells.
 16. A compositionfor use in killing of disease target cells comprising:a) a quantity ofleukocyte effector cells that express Fc receptors for antibody, andthat have been activated by combining them in vitro with interleukin-2for a sufficient period of time to increase the ADCC activity of saidquantity of effector cells; and b) a monoclonal antibody which iscapable of ADCC mediation and which primarily immunoreacts with anantigen that is specific to the disease target cell or that is presentin higher densities on the disease target cell than on normal cells. 17.A composition for use in killing of disease target cells comprising:a) aquantity of leukocyte effector cells that express Fc receptors forantibody, and that have been activated by combining them in vitro withinterleukin-2 for a sufficient period of time to increase the ADCCactivity of said quantity of effector cells; and b) a polyclonalantibody preparation that is capable of ADCC mediation and whichselectively immunoreacts with the disease target cell or with an antigenthat is present in higher densities on the disease target cell than onnormal cells, wherein such composition has cytotoxic effect specificallyon such disease target cells.